Nano-flares are a new class of intracellular molecular probes.
This blog is only concerned with “nano-flares” that are used as molecular probes. Hence it will only deal with answer 2.
Illustration of a nano-flare probe
Several recent papers report the use of nano-flares as novel and potentially very useful probes. These types of probes combine several unique features. Nano-flare probes allow for the transfection of a cell and the detection and quantification of RNA molecules in living cells. Nano-flare probes are also protected against enzymatic degradation.
The use of these nano-flare probes in siRNA knockdown experiments has been demonstrated but it is reported that nano-flares will also be very useful in other areas such as cell sorting, gene profiling, and real-time drug validation studies. In addition, the unique features of these new materials may also be very useful as gene regulation agents and they may be easily adapted to allow for the simultaneous transfection, control, and visualization of gene expression in real time.
How do these probes work?
Gold nanoparticles are functionalized with a recognition sequence which is hybridized with a short complementary Cy5 labeled reporter strand called a nano-flare, which is capable of being displaced by the target oligonucleotide. After the nano-flare nanoprobes enter the cell, the reporter “Flare” is displaced by the specific incoming RNA molecule present in the cell. The release of the flare results in a fluorescent signal. The intensity of the signal corresponds with the relative amount of the target RNA in the cell.
Seferos et al. in 2007 report the design and use of functionalized nanoparticles as probes to transfect living cells and to detect mRNA in these cells as well. The researchers demonstrated that these probes can be used to provide a fluorescence signal that correlates with the relative amount of a specific intracellular RNA. Furthermore, the scientists report that these new probes do not require microinjection or transfection reagents to enter the cells. The probes are highly resistant to enzymatic degradation and are non-toxic under the conditions used by the researchers. The paper shows that because of the unique properties of these probes they can be used to quantify RNA levels and are less prone to nonspecific signaling in comparison to the use of free oligonucleotide probes. The probes were designed using 13 nm gold nanoparticles that can be densely functionalized with thiolated oligonucleotides or oligonucleotide mimics such as oligonucleotide chimers using bridged nucleic acids (BNAs) to increase their stability and specificity.
Prigodich et al. in 2009 reported the use of nano-flares for mRNA regulation and detection. The researchers report the characterization of the binding rates and specificity of nano-flares using the gene survivin as a target. This gene is associated with the diagnosis and treatment of cancer. DNA-BNA (LNA) chimeras were used for the preparation of the nano-flares to increase mRNA binding affinities. The goal was to increase the detection and regulatory efficiency of the probes. Furthermore the researchers reported that after the nano-flares enter the cells they bind mRNA which results in a decrease of the relative abundance of mRNA and a fluorescent response in a dose- and sequence-dependant manner. The researchers argue that this work is important for the development of an mRNA responsive ‘theranostic’.
Theranostics (A term made up by combining the two terms therapeutics and diagnostics) is a recently proposed process of diagnostic therapy for individual patients that involves testing them for possible reaction to a new medication and to tailor a treatment for them based on the test results.
In 2009 Zheng et al. published a paper in which they report the development of a nucleic acid aptamer nano-flare probe that has a high affinity for adenosine triphosphate (ATP) allowing the molecular detection of this small molecule in living cells.
Next, Prigodich et al. in 2012 reported the development of a multiplexed nano-flare. This nanoparticle flare allows to simultaneously detect two distinct mRNA targets inside a living cell. The scientists report that one of the targets can be used as an internal control. This feature improves the detection of mRNAs by accounting for cell to-cell variations in nanoparticle uptake and background. The researchers argue that these structures allow the user to determine more precisely relative mRNA levels in individual cells and that this will improve cell sorting and quantification. Furthermore, this type of nano-flares allow for the detection of mRNA in living cells and to measure relative mRNA levels. It is thought that the ability to do this will increase our knowledge in understanding of fundamental cellular events in biology and diseases. Multiplexed nano-flares add the ability to detect multiple targets at once and can act as internal controls for a cell to cell comparison by measuring the mRNAs for a gene of interest in comparison to the mRNA of a second gene.
The scientists conclude that multiplexed nano-flares allow the detection of multiple targets in a single experiment, have the potential for the separation of cell types based upon internal targets, and allow for the relative quantification of mRNA levels within live cells.
Reported sequences used for the preparation of nano-flare probes
|1: Seferos et al., 2007:||DNA based nano-flare.|
|Recognition sequenceHomo sapiens survivin variant 3 alpha mRNA||5’-CTT GAG AAA GGG CTG CCA AAA AA-SH-3’|
|Reporter sequence||3’-CCC GAC GGT T-Cy5-5’|
|Target sequenceSurvivin||3’-GAA CTC TTT CCC GAC GGT-5’|
|Control particle recognition||5’-CTA TCG CGT ACA ATC TGC AAA AA-SH-3’|
|Control particle reporter||5’-Cy5-TGC AGA TTG TAC G-3|
|Survivin molecular beacon||5’-(Cy5)-CGA CGG AGA AAG GGC TGC CAC GTC G dabcyl-3’|
|Control molecular beacon||5’-(Cy5)-CGA CGT CGC GTA CAA TCT GCC GTC G-dabclyl-3’|
|2: Prigodich et al. 2009:||BNA/DNA chimera based nano-flare (LNA).|
|Target DNA||5′-CAA GGA GCT GGA AGG CTG GG – 3′|
|Survivin||5′-CCC AGC CTT CCA GCT CCT TG – (A)10 – propylthiol – 3′; N = BNA|
|Control||5′-TCT CCC CAG CCA GCT CCT TG – (A)10 – propylthiol – 3′; N = BNA|
|Flare||5′-(Cy5)-TCA AGG AGC TGG – 3′|
|Particle used in selectivityexperiments||5′-GCT TGC TTT GTG ATC ATA CC (A)10- propylthiol – 3′|
|Full complement||5′-GGT ATG ATC ACA AAG CAA GC-3′|
|Single mismatch||5′-GGT ATC ATC ACA AAG CAA GC-3′|
|Two mismatch||5’-GGT ATC ATC ACA AAG GAA GC-3′|
|Three mismatch||5′-GGT ATC ATC AGA AAG GAA GC-3′|
|Four mismatch||5′-GCT ATC ATC AGA AAG GAA GC-3′|
|3: Zheng et al. 2009:||Aptamer nano-flares; Synthesized from citrate-capped 13-nm Au NP precursors.|
|Thiol terminated ATP binding aptamers||5’-ACC TGG GGG AGT ATT GCG GAG GAA GGT GTC ACA (A)10 propyl thiol -3’|
|fluorophore-labeled (Cy5) reporter sequences||5’-(Cy5)-TGT GAC ACC TTC CT -3’|
|4: Prigodich et al. 2012:||Development of a multiplexed nano-flare.|
|Survivin Target||5’-CAA GGA GCT GGA AGG CTG GG-3’|
|Survivin Thiol||5’-CCC AGC CTT CCA GCT CCT TG –(A)5-PROPYLTHIOL-3’|
|Survivin Cy5 Flare||5’-(Cy5)-TCA AGG AGC TGG-3’|
|Survivin Cy3 Flare||5’-(Cy3)-TCA AGG AGC TGG-3’|
|Actin Target||5’-GGT CGC AAT GGA AGA AG—3’|
|Actin Thiol||5’-CTT CTT CCA TTG CGA CC-(A)5- PROPYLTHIOL-3’|
|Actin Cy5 Flare||5’-(Cy5)-TGG TCG CAA TGG-3’|
|Actin Cy3 Flare||5’-(Cy3)-TGG TCG CAA TGG-3’|
How are these probes prepared?
Nano-flare probes can be prepared using published procedures. Citrate-stabilized gold nanoparticles (13 ± 1 nm) are used and thiol-modified oligonucleotides are added to 13 ± 1 nm gold colloids at a concentration of 3 nmol of oligonucleotide per 1 mL of 10 nM colloid and shaken overnight. After 12 hours, a sodium dodecylsulfate (SDS) solution (10%) is added to the mixture to achieve a 0.1 % SDS concentration. Phosphate buffer (0.1 M; pH = 7.4) is added to the mixture to achieve a 0.01 M phosphate concentration, and six aliquots of sodium chloride solution (2.0 M) are added to the mixture over an eight-hour period to achieve a final sodium chloride concentration of 0.15 M. The mixture is shaken overnight to complete the functionalization process.
Next, the solution containing the functionalized particles are centrifuged (13,000 rpm, 20 min) and resuspended in phosphate buffered saline (PBS; 137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, pH 7.4) three times to produce the purified Au NPs.
The concentration of the particles can be determined by measuring their extinction at 524 nm (ε = 2.7 x 108 L mol-1 cm-1). Finally, the purified oligonucleotide functionalized Au NPs can be suspended to a concentration of 10 nM in PBS containing 100 nM of the complementary Cy5 labeled reporter sequence. To hybridize the reporter flare to the nano-particles the mixture is heated to 70° C and slowly cooled to room temperature. The resulting particles can now be stored in the dark for at least 12 hours to allow the hybridization to occur and the particles can be filter sterilized using a 0.2 μm acetate syringe filter.
Oligonucleotides can be synthesized using an automated oligonucleotide synthesizer such as the Expedite 8909 Nucleotide Synthesis System (ABI) or a similar system using standard solid-phase phosphoramidite chemistry. Bases and reagents needed are commercially available and the resulting oligonucleotides can be purified by reverse-phase high performance liquid chromatography (HPLC).
Chen T, Wu CS, Jimenez E, Zhu Z, Dajac JG, You M, Han D, Zhang X, Tan W. DNA micelle flares for intracellular mRNA imaging and gene therapy. Angew Chem Int Ed Engl. 2013 Feb 11;52(7):2012-6. doi: 10.1002/anie.201209440. Epub 2013 Jan 14. PubMed PMID: 23319350.
Frens, G.; “Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions”, Nature (London), Phys. Sci. 1973, 241, 20-22.
Prigodich AE, Seferos DS, Massich MD, Giljohann DA, Lane BC, Mirkin CA. Nano-flares for mRNA regulation and detection. ACS Nano. 2009 Aug 25;3(8):2147-52. doi: 10.1021/nn9003814. PubMed PMID: 19702321; PubMed Central PMCID: PMC2742376.
Seferos DS, Giljohann DA, Hill HD, Prigodich AE, Mirkin CA. Nano-flares: probes for transfection and mRNA detection in living cells. J Am Chem Soc. 2007, Dec 19;129(50):15477-9. Epub 2007 Nov 23. PubMed PMID: 18034495; PubMed Central. PMCID: PMC3200543.
Wu C, Chen T, Han D, You M, Peng L, Cansiz S, Zhu G, Li C, Xiong X, Jimenez E, Yang CJ, Tan W. Engineering of switchable aptamer micelle flares for molecular imaging in living cells. ACS Nano. 2013 Jul 23;7(7):5724-31. doi: 10.1021/nn402517v. Epub 2013 Jun 14. PubMed PMID: 23746078.
Zheng D, Seferos DS, Giljohann DA, Patel PC, Mirkin CA. Aptamer nano-flares for molecular detection in living cells. Nano Lett. 2009 Sep;9(9):3258-61. doi: 10.1021/nl901517b. PubMed PMID: 19645478; PubMed Central PMCID: PMC3200529.
Structures of bridged nucleic acids (BNAs)
has 28 years experience in the analysis and synthesis of natural and synthetic peptides, proteins, DNA and RNA oligonucleotides, bioconjugates, DNA and RNA probes and other biomolecules of interest.
BSI can help you with your probe design.
You can call us at 1-800-227-0627
or visit our website at
We are here to help you with all your scientific research needs!
Categories: Artificial Nucleic Acids, Bioanalysis, BNA RNA, BNAs, Bridged Nucleic Acid, Bridged Nucleic Acids, cell culture, Cellular Reprogramming, conjugation, DNA Hybridization, DNA-BNA chimeras, DNA-LNA chimeras, Gene Expression, Hybridization, miRNA, Molecular Probes, nano-flare probe, non-coding RNAs, Regulatory RNA, RNA, RNA Editing, RNAi, siRNA knockdown, Theranostics, Virus